Goat anti mouse iga hrp

Anti-spike IgA titers were also measured by indirect ELISA. Specifically, 96-well high-binding ELISA plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 100 μL of 0.8 μg/mL (80 ng/well) of Spike protein diluted in PBS. After washing plates with PBS-T, wells were blocked with 200 μL 3% Skim milk (Thermo Fisher Scientific) in PBS and incubated at 37 °C for 2 h and washed with PBS-T. 100 μL of sample was loaded on the plates using half-log serial dilution (equivalent to 3.162 × serial dilution starting at 1:10) and incubated at 37 °C for 1 h. Plates were washed again with PBS-T and 100 μL of goat anti-mouse IgA-HRP (Abcam, Cambridge, UK) diluted 1:10,000 added to each well. Plates were incubated at 37 °C for 45 min and washed with PBS-T. 100 μL of TMB substrate Sure-blue reserve one-component (Mandel Scientific Company Inc., Guelph, ON, Canada) was then added to each well and reaction stopped after 10 min incubation at room temperature by adding 100 μL of TMB stop solution one-component (Mandel Scientific Company Inc., Guelph, ON, Canada). Bound IgA Abs were detected spectrophotometrically at 450 nm, and IgA antibody titers were determined as above for IgG.

Indirect ELISA: Inactivated type-O FMDV from the FMD type-O liquid-phase blocking ELISA (LPBE) antibody detection kit was used as coating antigen on ELISA plates (Costar), and the free binding sites were blocked with 5% skim milk in PBS-Tween-20 (PBST). Mucosal samples to be tested and goat anti-mouse IgA (HRP; Abcam) were added in sequence. TMB was used as substrate to detect antibody responses. After the reaction was stopped, the optical density (OD) was measured at 450 nm. The contents of different types of IgG in serum at 28 days after immunization were detected by indirect ELISA. The ELISA plate was coated with inactivated type-O FMDV overnight at 4°C, blocked with 5% skim milk in PBST, and serum after immunization for 28 days (diluted 100 times) and goat anti-mouse IgG1, IgG2a (HRP; Abcam) were added. TMB was used as substrate and the plate was read at 450 nm.
Liquid phase blocking ELISA: Orbital venous blood of immunized mice was collected and the serum was isolated by centrifugation. Specific antibodies in the serum of immunized mice were detected and analyzed using the FMD type-O LPBE antibody detection kit (Lanzhou Shouyan Biotechnology Co., Ltd.).
Double sandwich ELISA: Serum interferon (IFN)-γ and interleukin (IL)-4 on day 28 after immunization were detected by mouse IFN-γ and IL-4 ELISA kits (Shenzhen Xin Bosheng Biological Technology Co., Ltd.).